Glenn P. Holley

Increased endothelial cell density in the paracentral and peripheral regionsof the human cornea

PURPOSE To systematically investigate the central, paracentral, and peripheral endothelial cell density (ECD) in normal human corneas. DESIGN Observational case series and experimental study. METHODS Noncontact specular microscopy was undertaken to determine the ECD of the central, paracentral (2.7 +/- 0.2 mm from center) and peripheral (4.7 +/- 0.2 mm from center) regions of the cornea of 48 normal eyes. The ECDs of central and peripheral regions were also determined with contact specular microscopy in 21 normal eyes and a group of 30 Optisol-GS eye bank corneas were evaluated with alizarin red stain. Histologic ECD of 13 Optisol-GS stored corneas were also determined. RESULTS Paracentral and peripheral ECD measured with the noncontact specular microscope were 5.8% (P <.01) and 9.6% (P <.001) increased compared with central ECD. Superior peripheral ECD was increased compared with the other three peripheral quadrants (P <.05) and was 15.9% higher than central ECD. Contact specular microscopy showed an increase of 8.9% in the peripheral ECD from the center. Alizarin red stained corneas confirmed the specular microscopy numbers with a 9.2% increase in the paracentral region, and a 17.2% increase in the peripheral region. Histological cross sections of human corneas also showed a 22.9% increase in peripheral ECD compared with the central region. CONCLUSIONS The human cornea has an increased ECD in the paracentral and peripheral regions of cornea compared with the central region. The superior peripheral region of the corneal endothelium has the largest increase in ECD. These data on normal endothelial cell distribution in the human cornea are especially significant as they relate to new surgical techniques and endothelial wound repair.

The effects of intraocular lidocaine on the corneal endothelium

OBJECTIVE The study aimed to evaluate the direct effect of intraocular lidocaine hydrochloride (HCl) 1% on corneal endothelial cell function, ultrastructure, and viability using an in vitro perfusion specular microscope system. DESIGN Paired rabbit and human corneas were isolated and mounted in an in vitro specular microscope for endothelial perfusion evaluation. Corneas were perfused with a control solution (BSS Plus for humans, glutathione bicarbonate Ringers [GBR] for rabbits) for a 1-hour stabilization period. After the stabilization period, one cornea of each matched pair was perfused with preservative-free lidocaine HCl 1% for 15 minutes followed by control solution for an additional 2 to 3 hours. The control cornea continued to receive either GBR or BSS Plus. Corneal thickness measurements were taken every 15 minutes throughout the perfusion period. Corneal swelling and deswelling rates were calculated by linear regression analysis. At the end of the experiment, corneas were fixed for scanning and transmission electron microscopy. In another group of corneas, the endothelial viability was assayed after direct perfusion with lidocaine HCl 1%. RESULTS Lidocaine HCl 1% caused endothelial cell edema, which reversed on removal of lidocaine from perfusion media. Corneal swelling and deswelling rates did not differ significantly between the lidocaine and control groups. Electron microscopy showed the effects of transient endothelial cell edema with an otherwise normal mosaic pattern and ultrastructure for both treatment groups. Endothelial cell viability was maintained after the direct lidocaine exposure and a 2-hour washout. CONCLUSIONS Lidocaine HCl 1% causes a transient endothelial cell edema to the in vitro perfused endothelium of human and rabbit corneas. Proper attention should be given to the type of lidocaine injected intraocularly (i.e., concentration, vehicle, preservatives, pH, osmolarity). Although lidocaine HCl 1% appears to be safe to both human and rabbit endothelium during short-term in vitro exposure, further in vivo and in vitro studies are needed to determine long-term effects of intraocular lidocaine on the corneal endothelium.

Effect of Mitomycin C on the Corneal Endothelium When Used for Corneal Subepithelial Haze Prophylaxis Following Photorefractive Keratectomy

PURPOSE To evaluate the potential effect of topical mitomycin C (MMC) on the corneal endothelium of myopic patients undergoing photorefractive keratectomy (PRK). METHODS Sixteen eyes with a planned ablation depth >75 microm underwent PRK followed by 0.02% MMC applied for 12 seconds using a methylcellulose sponge. Endothelial specular microscopy was performed with the Keeler-Konan specular photomicroscope in 16 eyes before and at least 1 year after surgery. Mean follow-up was 18 months (range: 12 to 24 months). Mean cell density, coefficient of variation of mean cell area, and percentage of hexagonal cells were measured and calculated using computerized morphometric analysis. RESULTS Mean endothelial cell densities before and after surgery were 2882 +/- 783 cells/mm2 (range: 1511 to 4022 cells/mm2) and 2867 +/- 588 cells/mm2 (range: 1638 to 3881 cells/mm2), respectively (P > .05). Mean coefficient of variation before and after surgery was 0.30 +/- 0.07 (range: 0.23 to 0.49) and 0.26 +/- 0.04 (range: 0.22 to 0.33), respectively (P=.06). Mean percentage of hexagonal cells before and after surgery was 61% +/- 6.8% (range: 47% to 70%) and 66% +/- 6.7% (range: 54% to 75%), respectively. CONCLUSIONS Administration of MMC for haze prophylaxis following PRK did not have a significant effect on quantitative endothelial cell density or qualitative morphometric parameters in this study.

Corneal Endothelial Cytoskeletal Changes in F-Actin With Aging, Diabetes, and After Cytochalasin Exposure

We investigated the changes in endothelial cytoskeletal F-actin that occur with aging, diabetes, and exposure to cytochalasin D. Rabbit corneas, human donor corneas (with or without polymegethism), and corneas of diabetic individuals were studied. Endothelial F-actin was stained using nitrobenzoxadiazole-phallacidin. Results of these experiments demonstrated that F-actin of the rabbit and human corneal endothelium was arranged in linear circumferential strands that formed a hexagonal array. After in vitro perfusion of cytochalasin D to the corneal endothelium, the F-actin became randomly distributed throughout the cytoplasm, the hexagonal shape of the endothelial cell was disrupted, and endothelial permeability to carboxyfluorescein increased. Changes in F-actin were also observed in the endothelium of the human corneas with polymegethism, and in donor tissue having had previous posterior chamber intraocular lens implantation. The corneas of diabetic individuals also showed marked irregular F-actin fibers crossing the endothelial cell cytoplasm. These abnormal patterns of F-actin may contribute in part to the polymegethism observed in the corneal endothelial cells and may be the result of constant stress in cell volume regulation, particularly in the corneas of diabetic individuals.

Effect of indocyanine green intraocular stain on human and rabbit corneal endothelial structure and viability an in vitro study

Purpose: To evaluate the direct effect of intraocular indocyanine green (ICG) on endothelial cell function, ultrastructure, and viability in human and rabbit corneas. Setting: A laboratory evaluation study. Methods: Paired human and rabbit corneas were mounted in an in vitro specular microscope for endothelial cell perfusion. One corneal endothelium was perfused with 25 mg ICG dissolved in 0.5 mL aqueous solvent in 4.5 mL balanced salt solution (BSS®) for 3 minutes followed by washout with a control solution. The percentage of ICG exposed to the corneal endothelium was 0.5%. The paired cornea was perfused with the same solution without ICG, followed by the washout. The corneas were fixed for scanning and transmission electron microscopy (TEM). In another group, the endothelial viability was determined using a live cell/dead cell assay. Results: In rabbit corneas, the mean corneal swelling rate was 12.9 &mgr;m/h ± 1.2 (SEM) in the ICG corneas and 2.8 ± 1.9 &mgr;m/h in the controls. Scanning electron microscopy and TEM revealed a normal endothelial cell mosaic. The control electron micrographs were similar. In human corneas, the mean swelling rate was 19.1 ± 2.8 &mgr;m/h in the ICG corneas and 19.2 ± 2.6 &mgr;m/h in the controls. Scanning electron microscopy and TEM revealed intact junctions with slight cellular vacuolization, similar to that in the controls. In the live cell/dead cell subgroup, the mean damage was 17.3% ± 1.7% in the ICG‐exposed corneas and 22.0% ± 8.9% in the controls. Conclusions: Three‐minute exposure to ICG in BSS had no adverse effect on corneal endothelial function, ultrastructure, or viability in human and rabbit corneas. This study provides a safety profile for the corneal endothelium when ICG is used as an intraocular tissue stain in ophthalmic surgery.

Corneal Endothelial Permeability of Human Tissue After Storage in Optisol

The purpose of this study was to compare Optisol to moist chamber storage for maintaining human corneal endothelial barrier function. Human corneas preserved in Optisol were stored for up to 35 days at 4 C. Endothelial carboxyfluorescein permeability (P(ac)) was measured and endothelial ultrastructure was evaluated by electron microscopy. Endothelial P(ac) (x 10(-4) cm/min) of Optisol-stored corneas was 1.7, 2.0, and 3.1 at five, seven, and 14 days, respectively. The P(ac) increased to 6.5 at 35 days of storage. Endothelial P(ac) in moist chamber stored-eyes was 2.6 at two days, and increased to 13.5 14 days of storage. Multiple regressional analysis showed that storage time and donor age affected P(ac); but time from death to enucleation, time from enucleation to storage, or endothelial cell number did not. Electron microscopy showed that endothelial junctions were maintained through two weeks by Optisol. Large areas of cellular destruction were seen after five days of moist chamber storage. These results show that Optisol can preserve endothelial barrier function through 14 days; barrier function is lost by three days of moist chamber storage.

Intraocular irrigating solutions: a comparison of Hartmann's lactated Ringer's solution, BSS and BSS Plus

Abstract• Background: We evaluated the effects of Hartmanns lactated Ringers (HLR) solution, balanced salt solution (BSS) and BSS Plus on human corneal endothelium. • Methods: Paired human corneas were mounted in the in vitro specular microscope for endothelial perfusion with HLR, BSS or BSS Plus for 15, 30, 60 and 120 min. Reversal experiments with BSS Plus after initial HLR perfusion were performed. At the end of the perfusions, electron microscopy, F-actin staining of the endothelial cytoskeleton and endothelial permeability measurements were carried out. • Results: Longterm perfusion (120 min) with HLR resulted in a significantly higher swelling rate than in the paired controls perfused with BSS Plus. Short-term exposure to HLR for 15, 30 and 60 min after initial BSS Plus perfusion increased the swelling rates significantly. The increased corneal swelling after HLR perfusion for 60 min was reversed by BSS Plus perfusion. Ultrastructural changes in HLR-perfused corneas included endothelial cell edema, cytoplasmic vacuolation and mitochondrial swelling. F-actin staining showed overall cytoskeletal disorganization after perfusion with HLR. Corneal endothelial permeability was higher for BSS Plus-perfused corneas than with HLR solution. • Conclusion: The results suggest that the clinically observed corneal clouding during irrigation with HLR is due to endothelial cell edema and decreased endothelial pump function. However, this increased corneal swelling is reversible by perfusion with BSS Plus.

Endothelial cell loss after 4 mm cataract surgery

Abstract To assess whether a 4 mm scleral tunnel incision with a 1.5 mm internal corneal lip (three‐step procedure) causes increased endothelial cell loss and damage to the cornea, we retrospectively evaluated the outcomes of 20 patients (40 eyes) who had a standard 4 mm scleral tunnel incision (two‐step procedure) in one eye followed by a three‐step incision in the second eye, with in situ phacoemulsification and insertion of a foldable silicone lens in each eye. Mean phacoemulsification time was 2.4 ± 1.1 minutes for the two‐step incisions and 3.4 ± 1.4 minutes for the three‐step incisions. Preoperative and postoperative endothelial cell counts were obtained to determine the effects of surgery on the corneal endothelium. Although the three‐step procedure had a trend toward increased endothelial cell loss from the central corneal region compared with the two‐step incision, the result was neither clinically nor statistically significant. The difference between the three‐step and two‐step incisions in postoperative endothelial cell counts from the superior corneal region was statistically significant. The difference in postoperative counts from the inferior region was not statistically significant. Although the three‐step 4 mm incision does seem to affect the corneal endothelium, its clinical significance is unknown.

Evaluation of endothelial mucin layer thickness after phacoemulsification with next generation ophthalmic irrigating solution.

Purpose: To evaluate human corneal endothelial mucin layer thickness and ultrastructure after phacoemulsification and irrigation-aspiration with either next generation ophthalmic irrigating solution (NGOIS) or BSS PLUS. Methods: Paired human corneas were mounted in an artificial anterior chamber, exposed to 3 minutes of continuous ultrasound (US) at 80% power using the Alcon SERIES 20000 LEGACY surgical system (n = 9) or to 2 minutes of pulsed US at 50% power, 50% of the time at 20 pps using the Alcon INFINITI Vision System (n = 5), and irrigated with 250 mL of either NGOIS or BSS PLUS. A control group of paired corneas did not undergo phacoemulsification or irrigation-aspiration (n = 5). Corneas were divided and fixed for mucin staining or transmission electron microscopy. Mucin layer thickness was measured on the transmission electron microscopy prints. Results: The mucin layer thickness in the continuous phaco group was 0.77 ± 0.02 μm (mean ± SE) with NGOIS and 0.51 ± 0.01 μm with BSS PLUS (t test, P < 0.001). The mucin layer thickness in the pulsed phaco group was 0.79 ± 0.02 μm with NGOIS and 0.54 ± 0.01 μm with BSS PLUS (P < 0.001). The mucin layer thickness in the untreated control group was 0.72 ± 0.02 μm. The endothelial ultrastructure was normal in all corneas. Conclusions: In this in vitro corneal model, NGOIS, due to its lower surface tension and higher viscosity, preserved endothelial mucin layer thickness better than BSS PLUS with both the INFINITI Vision System (pulsed US) and the LEGACY surgical system (continuous US).

Corneal Edema from an Intraocular Irrigating Solution Containing Benzalkonium Chloride

AbstractAn intraocular irrigating solution developed in India (Irrisol) has been reported to cause immediate corneal edema when used during cataract surgery. The purpose of this study was to evaluate this intraocular irrigating solution for endothelial toxicity and compare it to balanced salt solution (BSS), a similar irrigating solution used in the United States. Human and rabbit corneas were mounted for in vitro specular microscopy and endothelium perfused with Irrisol and BSS.The results of this study showed that Irrisol caused immediate corneal swelling, with marked destruction of the corneal endothelial cells when observed with scanning and transmission electron microscopy. Further analysis showed that the Irrisol had a lower surface tension (35 dynes/cm) compared to BSS (73 dynes/cm). High-performance liquid chromatographic (HPLC) analysis confirmed that the lower surface tension and the endothelial damage were due to the addition of benzalkonium chloride (BAC) (0.009%) in the Irrisol intraocular ir...